Mammalian blood contains small cells known as platelets. Human platelets are anucleate circulating particles with a diameter of about 2-4 micrometers. Platelets that circulate freely through blood vessels as free floating cells are known as "resting" platelets.
Intact, resting platelets are normally non-adhesive and exhibit limited interaction with intact endothelium or with other blood cells. However, these platelets are rapidly converted from free floating cells to an adhesive aggregate mass of intact platelets, known as "activated platelets", during the process of hemostasis or during the formation of thrombi.
When the wall of a blood vessel is damaged as from a blow or as a result of vascular event, a hemostatic response is rapidly elicited to prevent excessive blood loss. This response, as it has evoIved in higher organisms, is triggered to efficiently seal and localize hemostasis by the concerted action of blood platelets and the coagulation system. In contrast, in vessels injured by chronic disaases; e.g., atherosclerosis, activated platelets may adhere to each other and to the vessel wall leading to thrombotic occlusion. Such events occur in heart attack and stroke with venous embolism. Blood platelets are transported to the site of the injury and aggregate to form a hemostatic plug. Fibrinogen then assists in forming a clot.
The involvement of blood platelets in primary hemostasis and thrombosis is initiated by surface contact interactions and is contingent upon the adhesive properties of the platelet cell. Three general phenomena comprise an adhesive event: (1) attachment of the platelet to the substratum, such as the subendothelial matrix; (2) spreading of the platelet which establishes additional contact sites with the substratum; and (3) recruitment of additional platelets to form aggregates. Platelet aggregation can be stimulated by exposure of the cell to adenosine-5'-diphosphate (ADP), ADP/fibrinogen, epinephrine, thrombin, serotonin, collagen and certain arachidonic acid metabolites.
The expression of adhesive properties in a resting human blood platelet therefore is initiated by an appropriate stimulus that regulates the adhesive response. The stimulation transforms blood platelets from a non-adhesive "resting" state to an adhesive "activated" state that supports adherence of the platelets to blood vessels and to one another to form thrombi. Thus, the alteration of the adhesive properties of these cells plays a crucial role in host defense mechanisms and in the pathogenesis of thromboembolic disease.
The terms "non-stimulated platelet" and "resting platelet" are used interchangeably herein to denote a normal free flowing blood platelet that has not been exposed to a physiological stimulus as previously described, and which is, therefore, in a "non-adhesive" or "intact" state. Conversely, the terms "stimulated platelet" and "activated platelet" are used interchangeably herein to denote a blood platelet that has been exposed to a physiological stimulus as described, and which is, therefore, in an "adhesive" state.
During hemostasis and thrombosis, resting blood platelets are known to change morphologically in shape and biochemically in cell surface membrane composition during aggregation and secretion. The platelets change in shape from disks to spheres with long pseudopods that aggregate with adjacent platelets. The activated platelets secrete their granule contents into the surrounding environment, accelerate plasma coagulation, bind developing fibrin strands and provide the impetus for clot retraction.
A study of platelet surface membrane changes following the stimulation of resting platelets with thrombin showed that major platelet surface membrane changes occur after secretion but not after reversible aggregation. See George et al., J. Clin. Invest., 66, 1 (1980) which is incorporated herein by reference.
Another study of the action of thrombin on intact human blood platelets related to the presence of a new cell membrane-associated protein that was not fibrinogen. Baenziger et al., Proc Natl. Acad. Sci. (U.S.A.), 68, 240 (1971) which is also incorporated herein by reference. This new membrane protein has since been identified as "thrombospondin" and is sometimes also known as "thrombin-sensitive protein" or "glycoprotein G".
Thrombospondin is a filamentous macromolecule measuring about 7.times.70 nanometers (nm) that appears to be a major subcellular blood protein component [Lawler et al., J. Biol. Chem., 23, 8609 (1978)]. Lawler et al. also reported that thrombospondin comprises three disulfide-linked chains of about 142,000 to about 190,000 daltons each which may or may not be identical. Thrombospondin contains approximately 4 percent by weight carbohydrate, with neutral sugars, amino sugars and sialic acid (N-acetylneuraminic acid) in a weight percent ratio of about 4:3:1.5, and contains no hexuronic acid.
From studies of patients with quantitative deficiencies of certain plasma proteins, platelet adhesivity is believed to be mediated or modulated by selected "adhesive proteins" in addition to the foregoing stimulus requirement.
Adhesive proteins are cofactor proteins that are expressed at the outer cell surface of an activated blood platelet These proteins are usually either completely absent from the outer cell surface of intact resting platelets or are present only in minimal quantities. Thus, cell membrane-associated adhesive proteins are believed to be primarily expressed as intracellular components, and in some instances as extracellular components.
Adhesive proteins that have been identified on the outer cell membrane surface of activated platelets include fibrinogen, fibronectin, von Willebrand factor (also known as Factor VIII) and thrombospondin. These four proteins share common characteristics in that they (1) are normally absent from the outer cell surface of resting platelets; (2) interact minimally with non-stimulated intact platelets; (3) are expressed at the cell surface following platelet stimulation and (4) have gross structural similarities. Proposed mechanisms for the expression of these four adhesive proteins at the outer cell membrane platelet surface are discussed in Ginsberg and Plow, J. Supramolecular Structure and Cellular Biochem., 17, 91 (1981).
Although the primary sequence homologies between the proteins are not fully known, selected structural features of the four proteins are similar. All four adhesive proteins described above, are glycosylated macromolecules having an element of internal symmetry in their multimeric state. In particular, fibrinogen has a molecular weight of about 340,000 daltons and has a dimeric structure made up of a plurality of subunits having molecular weights of about 48,000, 56,000 and 65,000 daltons. Fibronectin has a molecular weight of about 450,000 daltons and circulates primarily as a dimer of two very similar, but not identical disulfide-linked subunits having a molecular weight of about 220,000 and 230,000 daltons, respectively. Higher multimers of fibronectin are known to occur in plasma and may predominate in the matrix. Thrombospondin exists as a trimer having a molecular weight of about 450,000 daltons with three disulfide-linked subunits of very similar sizes having a molecular weight of from about 142,000 to about 190,000 daltons. von Willebrand Factor is composed of 220,000 dalton subunits that multimerize to yield a molecular weight of about 1,000,000 to about 20,000,000 daltons.
Plasma and extracellular matrices are believed to constitute the primary exogenous sources of the above four adhesive proteins. With the exception of thrombospondin, plasma is the major potential source of the other three adhesive proteins. Saglio et al. have reported thrombospondin to be 3 to 5 orders of magnitude less abundant than fibronectin or fibrinogen at a concentration of 20-130 nanograms (ng)/milliliter (ml) in blood plasma [Blood, 59, 162 (1982)].
It is known that, upon exposure to thrombin, blood platelets mobilize certain submembranous cytoplasmic storage granules (alpha granules) that release their contents into the environment. Platelet storage granules contain a number of proteins that are normally absent from the cell surface of a resting blood platelet, but that are present as cell surface associated membrane proteins of thrombin-stimulated "activated" platelets. It is further believed that the adhesive proteins, especially thrombospondin, reside primarily in endogenous pools within the alpha granules of the blood platelets.
Expression of the adhesive proteins on the platelet cell surface is dependent upon platelet stimulation that induces the secretion and release of alpha granule components. This release liberates the adhesive proteins into the extracellular milieu and provides their appearance on the cell surface. Additionally, since adhesive proteins are normally minimally associated with the surface of resting platelets, it is believed that the mechanisms for cell surface expression of adhesive proteins from exogenous sources are also initiated and dependent upon platelet stimulation.
Since platelet activation and localization are fundamental events of injuries to blood vessels, there is a need for methods and reagents capable of detecting and discriminating between activated blood platelets and resting platelets. The ability to detect activated platelets that may react selectively with impaired blood vessel walls would particularly aid in finding the situs of soft tissue injuries, especially in cases of trauma where the victim has no externally apparent injury.
Presently, assays of circulating activated platelets are measured in vitro using the well known platelet aggregate ratio test wherein appropriate dilutions of an unknown antibody-containing sample are incubated with antigen-bearing cells and the antibody concentration (titer) is determined by comparing the resulting agglutination responses to those obtained with a standard antibody-containing sample of known titer. This test, however, is often cumbersome and unreliable.
One current method for imaging blood platelets in vivo involves a time-consuming and inconvenient procedure using .sup.111 indium-labelled platelets. The procedure involves isolating blood platelets from a patient, labelling the platelets with the radionucleotide in vitro and then reinfusing the radiolobelled platelets back into the patient. The .sup.111 indium-labelled platelets can be imaged only after a sufficient period of time passes to reduce background signal interferences.
Thus, a diagnostic site-specific imaging reagent capable of selectively binding and imaging a specific cell membrane-associated adhesive protein would have desirable clinical utility for rapidly indicating the site of stimulated activated platelets and could be useful for discriminating between such platelets and resting platelets.
More partcularly, a diagnostic site-specific imaging reagent comprising an antibody or antibody fragment that binds to a specific blood platelet protein component (when it is expressed as a cell surface antigen of a blood platelet that is in a stimulated activated state) could circumvent many of the technical complexities, inconveniences and time consuming aspects of the presently available in vitro and in vivo diagnostic procedures.
Nieuwenhuis et al. reported a monoclonal antibody against thrombin-activated platelets that does not cross-react with resting platelets [Thrombosis and Haemostasis, 50, 100 (1983)]. The monoclonal antibody was prepared against activated platelets using spleen cells from mice immunized with thrombin-activated human blood platelets fused to a murine (mouse) myeloma cell line to form a hybridoma. Anti-platelet antibodies were produced, and one hybridoma produced antibodies that bound to an unknown antigen on thrombin- or ADP-activated platelets. However, the antibody had no functional properties in aggregation studies of platelets stimulated by ADP, epinephrine, collagen, thrombin or the antibiotic, restocetin. The antigen to which the antibody bound was found to be located in a special subclass of blood platelet granules different from alpha granules.
Hsu-Lin et al. also reported the production of monoclonal antibodies specific for thrombin-activated platelets [Blood, 62 (Suppl. 1), (1983)] prepared from murine hybridomas using standard methods known in the art. The specificity of the antibodies was to an unidentified single protein component that migrated between glycoprotein IIb and glycoprotein IIIa on separation by Western blotting procedures. The unidentified protein was derived from either solubilized activated platelets or from resting platelets, and had an apparent molecular weight of between about 98,000 and 120,000 daltons.
McEver et al. reported that a monoclonal antibody to platelet membrane glycoprotein IIa binds only to activated platelets [Blood, 62 (Suppl 1), (1983)]. The hybridoma was produced from spleen cells of a mouse immunized to thrombin-activated human platelets bound specifically to thrombin-activated platelets but not to peripheral blood mononuclear cells. The specificity of the antibody was to a protein having an apparent molecular weight of about 138,000 (unreduced) and 148,000 (reduced) daltons and fit certain criteria for glycoprotein IIa, which is believed to be a plasma membrane molecule that changes its conformation after platelet activation, or for a granule membrane component that fuses with the plasma membrane during the release reaction. However, the function of the protein is not known.
Thus, a need exists for a pre-labelled antibody to a blood protein component, particularly one located in the subcellular alpha granules capable of indicating the blood component as an expressed cell surface antigen by imaging the site as a means for detecting and localizing the blood component. An indicating site-specific diagnostic reagent could provide a rapid clinical evaluation means in scanning for platelet thrombi.
A particular need exists for a non-invasive rapid clinical method for identifying and localizing the presence of thrombi, especially in heart attack and stroke patients, and for managing in situ thrombolytic therapy in patients having acute myocardial infarction.
A need also exists for detecting in vivo the situs of injured blood vessel walls following an injury or to screen for silent coronary artery disease in individuals at risk.